SEETU GULIAMSC BIOTECHNOLOGYCENTRAL UNIVERSITY OF HARYANA

Genetic Model Compatible with Ig StructureA. Two models for Ab structure diversity1. Germ-line theory: maintained that theI. genome contributed by the germ cells, egg

and sperm, contains a large repertoire of immunoglobulin genes2. Somatic-variation theory: maintained thatthe genome contains a small number of

immunoglobulin genes, from which a large number of Ab specificities are generated in the somatic cells by mutation or recombination

B. The Two-Gene Model of Dreyer and Bennett- said that two separate genes encode a single immunoglobulin heavy or light chain,

one gene for the V region (variable region) and

the other for the C region (constant region)- these two genes must come together at

the DNA level to form a continuous message that can be transcribed and translated into a single Ig heavy or light chain

- their theory was essentially proven correct in time

C. Verification of the Dreyer and BennettHypothesis

- S. Tonegawa and N. Hozumi found that separate genes encode the V and C regions of Ig’s and that the genes are rearranged in the course of B-cell differentiation

- results prove the two-gene model is correct:one gene encodes the V region and

one encodes the C region

II. Multigene Organization of Ig Genes- germ-line DNA contains several coding sequences, called gene segments, separated noncoding regions

- gene segments are rearranged during B cell maturation to form functional Ig genes

by

- K and lambda light-chain families contain V, J,and C segments; the rearranged VJ segments encode the variable region of the light chains

- the heavy-chain family contains V, D, J, and C gene segments; the rearranged VDJ gene segments encode the variable region of the heavy chain- each V gene segment is preceded at its 5’ end by a signal or leader (L) peptide that guides the heavy or light chain through the endoplasmic reticulum- signal peptide is cleaved from the nascent light and heavy chains before assembly of the final Ig molecule

A. lambda-chain multigene family- functional lambda variable-region gene contains two coding segments – a 5’ V segment and a 3’ J segment – which are separated by a noncoding

DNA sequence in unrearranged germ-line DNA

- Mouse:- has two V-lambda gene segments, four J- lambda gene segments, and four C-

lambda gene segments- J-lambda4 and C-lambda4 are pseudogenes, which are defective

genes that are incapable of encoding protein

B. K-chain Multigene Family- in the mouse, the V-K and J-K gene

segments encode the variable region of the K light chain, and the C-K gene segment encodes the constant region

C. Heavy-chain Multigene Family-the V-H,D-H and J-H segments

encode the variable region of heavy chain , and the C-H gene segment encodes the constant region

III. Variable-Region Gene Rearrangements- this mechanism produces mature, immunocompetent B cells; each such cell is committed to produce antibody with a binding site encoded by the particular sequence of its rearranged V genes

A. V-J Rearrangements in Light-Chain DNA- in humans, any of the functional V-lambda genes can combine with any of the four functional J-lambda-C-lambda combinations

- in human and mouse K light-chain DNA,any one of the V-K gene segments can be

joined with any one of the functional J-K genesegments- for Kappa light-chain gene

rearrangement and RNA processing events required to generate a K light-chain protein

B. V-D-J Rearrangements in Heavy-ChainDNA

IV. Mechanism of Variable-Region DNA Rearrangements

A. Recombination Signal Sequences- flank each germ-line V, D, and J gene

segment- function as signals for the

recombination process that rearranges the genes

B. Enzymatic Joining of Gene Segments- V(D)J recombinase: catalyzes V-(D)-J recombination, which takes place at the junctions between RSSs and coding

sequences- recombination-activating genes (RAG-1

and RAG-2) encode for proteins that act synergistically to mediate V-(D)-J joining

- recombination of variable-region gene segments is a multistep process catalyzed by a system of recombinase enzymes

C. Defects in Ig-Gene Rearrangements- knockout mice lacking either RAG-1 or RAG-2 are unable to initiate the

recombination process because they cannot introduce

double- strand DNA breaks between the RSSs and coding sequences in germ-line

immunoglobulin DNA

- result: V, D, and J gene segments remain unrearranged

- since both B and T cells utilize the same recombination machinery, the RAG-

1- and RAG-2- knockout mice lack mature T and B cells and consequently exhibit a severe combined immunodeficiency (SCID)

D. Productive and NonproductiveRearrangements

- nonproductive rearrangement: imprecisejoining that results in gene segments that arejoined out of phase, so that the triplet reading frame for translation is not preserved.

- resulting VJ or VDJ unit will contain

numerous stop codons, which interrupt

translation

- Productive rearrangement: theresulting VJ or VDJ unit can be translatedin its entirety, which will yield a complete antibody

- these two type of rearrangements that occur produce only about 8% of pre-B

cells in the bone marrow that undergo maturation and leave as mature, immunocompetent B cells

E. Allelic Exclusion- even though a B cell is diploid, it expresses the rearranged heavy-chain genes from only

one chromosome and the rearranged light-chaingenes from only one chromosome.

known as allelic exclusionThis is

- this ensures that functional B cells nevercontain more than one VDJ from the heavy chain and one VJ unit from the light chain

V. Generation of Antibody DiversityA. Multiple Germ-Line V, D, and J GeneSegments

- contribute to the diversity of the antigen- binding sites in antibodies

B. Combinatorial V-J and V-D-J Joining- in humans, the ability of any of the 51 Vh

gene segments to combine with any of the 27Dh segments and any of the 6 Jh

segmentsallows a considerable amount of heavy-

chain gene diversity to be generated

C. Junctional Flexibility- increases the enormous diversity generated by means of V, D, and

J combinations- can lead to many nonproductive

rearrangements, but it also generates several productive combinations that encode alternative amino acids at each coding joint, thereby increasing antibody diversity

D. P-AdditionE. N-Addition

F. Somatic Hypermutation- results in additional antibody diversity

that is generated in rearranged variable- region gene units

- also causes individual nucleotides in VJ or VDJ units to be replaced with alternatives, thus possibly altering the specificity of the encoded immunoglobulins

- usually occurs within germinal centers- targeted to rearranged V-regions located within a DNA sequence containing about

1500 nucleotides, which includes the whole of the

VJ or VDJ segment

- largely random- affinity maturation: following exposure

to

antigen, those B cells with higher affinity receptors will be for survival because of their greater ability to biselected end to the antigen

- this takes place in germinal centers

G. Association of Heavy and Light Chains- combinational association of H and Lchains also can generate antibody diversity

VI. Class Switching Among Constant-RegionGenes- class switching: after antigenic stimulation of a

B cell the heavy chain DNA can undergo afurther rearrangement in which the VhDhJh unit can combine with any Ch gene segment

- switch region: DNA flanking sequenceslocated 2-3 kb upstream from each Ch

segment that are involved in class Switching-class switching depends on the interplay of four elements : switch regions , a switch recombinase , the cytokine signal that dictate the isotype to which B cell switches, and the enzyme called activation-induce cytidine deaminase

VII. Expression of Ig GenesA. Differential RNA Processing ofChain Primary Transcripts

Heavy-

- this can yield different mRNA’s- this processing explains the

production of secreted or membrane- bound forms of a particular immunoglobulin and the simultaneous expression of IgM and IgD by a single cell

B

1. Expression of Membrane or SecretedImmunoglobulin

- difference depends on differential processing of a common primary transcript

- mature naïvemembrane-bound

- differentiated

B cells produce onlyantibodyplasma cells produce

secreted antibodies

2. Simultaneous Expression of IgM and IgD- controlled by differential RNA processing- if the heavy chain transcript is cleaved and polyadenylated at site 2 after the Cu exons, then

the mRNA will encode the membrane form of the u

heavy chain- if polyadenylation is instead further downstream

at site 4, after the C delta exons, then RNA splicing

will remove the intervening Cu exons and produce

mRNA encoding the membrane form of the delta heavy

chain

B. Synthesis, Assembly, and SecretionImmunoglobulin

- heavy and light chains are

of

synthesized on separate polyribosomes ofthe rough ER

VIII. Regulation of Ig-Gene Transcription- three regulatory sequences in DNA regulate transcription of immunoglobulin genes:

1. promoters: promote initiation of RNAtranscription in a specific direction

- located about 200 bp upstream from transcription initiation site

2. Enhancers: nucleotide sequences located some distance upstream or downstream from a gene that activate transcription from the promoter sequence in an orientation-independent manner3. Silencers: nucleotide sequences that down-regulate transcription, operating in both directions over a distance

- each VH and VL gene segment has a promoter located just upstream from the leader sequence- these promoters contain a TATA Box

A. Effect of DNA Rearrangement onTranscription

- rate of transcription of a rearranged VlJl or VhDhJh unit is as much as 10,000X the rate

of transcription of unrearranged Vl or Vh

segments- oncogenes: genes that promote cellular proliferation or prohibit cell death

- these can often translocate to the immunoglobulin heavy or light chain loci

B. Inhibition of Ig-Gene Expression in Tcells

-and

-

complete Ig-gene rearrangement of HL chains occurs only in B cellscomplete TCR-gene rearrangement is

limited to T cells

SUMMARY

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